PROJECT SUMMARY The long-term goal of this research is to understand how chemokines recognize their binding partners in order to develop new molecules that alter chemokine signaling for therapeutic benefit. Chemokines and their cell- surface receptors form a network of signaling proteins that orchestrate the development and function of the cellular immune system by guiding the migration of white blood cells and homing of stem cells. Many of these proteins have been validated as drug targets for inflammatory and autoimmune diseases, HIV-AIDS, cardiovascular disease and cancer. Our previous work revealed the structural basis and functional importance of self-association, glycosaminoglycan binding, and receptor sulfotyrosine recognition for the chemokine CXCL12 and its receptor CXCR4, and demonstrated that engineered CXCL12 variants can be used to block cancer progression in animal models of metastatic disease. Atomic resolution details of other chemokine- receptor complexes are needed to guide the development of new small molecule and biologic drugs. With 46 chemokine ligands and 23 receptors, the human chemokine network represents the largest GPCR family with respect to the number of endogenous ligands and receptors. Across the chemokine family, ligand-receptor specificity varies from strictly monogamous to highly promiscuous, but the molecular determinants of selectivity are unknown. Until recently, details of the full chemokine-receptor interface were lacking due to the extreme challenges presented by crystallization of active GPCR complexes. However, the availability of a rich sequence database and multiple structures of chemokine-receptor complexes reported since the last renewal of this R01 now enable us to address the most pressing question in the chemokine field: how is selective promiscuity embedded in a collection of highly conserved chemokine and receptor structures? In the first specific aim of this competing renewal application we propose a comprehensive analysis of this complex signaling network, in order to decipher the ?chemokine code? governing receptor-ligand selectivity and promiscuity. The second aim will test the hypothesis that selective chemokine promiscuity is encoded across a broad protein-protein interface using the chemokines CXCL11 and CXCL12 and their receptors ACKR3, CXCR3 and CXCR4 as a model system to test the validity of the hierarchical model developed in aim 1. Aim 3 tackles the puzzle of extreme promiscuity in the chemokine family with the atypical receptor ACKR1 as an experimental testbed. ACKR1/DARC is a scavenger receptor on red blood cells that binds many different chemokines but also plays an essential role in hematopoiesis in the bone marrow. We hypothesize that ACKR1 uses an elongated disordered N-terminal domain to bind many protein ligands with distinct but overlapping binding sites. Completion of the work proposed here will provide a new foundation upon which the molecular determinants of chemokine-receptor recognition can be precisely and systematically elucidated.